Class III antiarrhythmic drugs block the rapid delayed rectifier K+ channel hERG and prolong ventricular repolarization, measured on the ECG as a lengthened QT interval. Drug-induced hERG blockade and QT prolongation also occurs as an unintended side effect of some common medications and increases the risk of torsades de pointes, a ventricular arrhythmia that can degenerate into ventricular fibrillation and sudden death. An understanding of the molecular determinants of drug binding to hERG channels would facilitate design of new drugs devoid of this dangerous side-effect. Treatment of congenital or drug-induced long QT syndrome is presently inadequate but a recently discovered hERG channel agonist represents a potential new therapy. The goals of this project are to characterize the mechanisms of action, and the structural basis of the binding site of hERG channel blockers and activators. We previously used an Ala-scanning mutagenesis approach to define the major structural determinants of the hERG channel that can account for block by high affinity blockers such as cisapride and terfenadine. Two aromatic residues in the S6 domain (Tyr652, Phe656) and three residues located at the base of the pore helix (Thr623, Ser624 and Val625) were required for potent block by structurally diverse drugs. In Aim 1, we will refine the molecular determinants for hERG block by high affinity ligands. Aim 2 will define the binding site for low affinity ligands (antibiotics) using site-directed mutagenesis and voltage clamp of mutant channels expressed in Xenopus oocytes. In Aim 3, we will determine the binding site and molecular basis of altered gating induced by two hERG channel activators, a fenamate and a novel quinolinylpiperidine.